This invention relates to the isolation, purification and characterization of proteins that influence the replication, differentiation or maturation of blood cells, especially platelet progenitor cells. This invention further relates to the cloning and expression of a protein ligand capable of binding to and activating mpl, a member of the cytokine receptor superfamily. This application further relates to the use of these proteins alone or in combination with other cytokines to treat thrombocytopenia.
I. Megakaryocytopoiesis
It is known that bone marrow pluripotent stem cells differentiate into megakaryocytic, erythrocytic, and myelocytic cell lines. It is believed there is also a line of committed cells between stem cells and megakaryocytes. The earliest recognizable member of the megakaryocyte (meg) family are the megakaryoblasts. These cells are initially 20 to 30 xcexcm in diameter having basophilic cytoplasm and a slightly irregular nucleus with loose, somewhat reticular chromatin and several nucleoli. Later, megakaryoblasts may contain up to 32 nuclei, but the cytoplasm remains sparse and immature. As maturation proceeds, the nucleus becomes more lobulate and pyknotic, the cytoplasm increases in quantity and becomes more acidophilic and granular. The most mature cells of this family may give the appearance of releasing platelets at their periphery. Normally, less than 10% of megakaryocytes are in the blast stage and more than 50% are mature. Arbitrary morphologic classifications commonly applied to the megakaryocyte series: are megakaryoblast for the earliest form; promegakaryocyte or basophilic megakaryocyte for the intermediate form; and mature (acidophilic, granular, or platelet-producing) megakaryocyte for the late forms. The mature megakaryocyte extends filaments of cytoplasm into sinusoidal spaces where they detach and fragment into individual platelets (Williams et al., Hematology, 1972).
Megakaryocytopoiesis is believed to involve several regulatory factors (Williams et al., Br. J. Haematol., 52:173 [1982] and Williams et al., J. Cell Physiol. 110:101 [1982]). The early level of megakaryocytopoiesis is postulated as being mitotic, concerned with cell proliferation and colony initiation from CFU-meg but is not affected by platelet count (Burstein et al., J. Cell Physiol. 109:333 [1981] and Kimura et al., Exp. Hematol. 13:1048 [1985]). The later stage of maturation is non-mitotic, involved with nuclear polyploidization and cytoplasmic maturation and is probably regulated in a feedback mechanism by peripheral platelet number (Odell et al., Blood 48:765 [1976] and Ebbe et al., Blood 32:787 [1968]). The existence of a distinct and specific megakaryocyte colony-stimulating factor (meg-CSF) has been disputed (Mazur, E., Exp. Hematol. 15:340-350 [1987]). Although. meg-CSF""s have been partly purified from experimentally produced thrombocytopenia (Hill et al., Exp. Hematol. 14:752 [1986]) and human embryonic kidney conditioned medium [CM] (McDonald et al., J. Lab. Clin. Med. 85:59 [1975]) and in man from aplastic anemia and idiopathic thrombocytopenic purpura urinary extracts (Kawakita et al., Blood 6:556 [1983]) and plasma (Hoffman et al., J. Clin. Invest. 75:1174 [1985]), their physiological function is as yet unknown in most cases. The conditioned medium of pokeweed mitogen-activated spleen cells (PWM-SpCM) and the murine myelomonocyte cell line WEHI-3 (WEHI-3CM) have been used as megakaryocyte potentiators. PWM-SpCM contains factors enhancing CFU-meg growth (Metcalf et al., Pro. Natl. Acad. Sci., USA 72:1744-1748 [1975]; Quesenberry et al., Blood 65:214 [1985]; and Iscove, N. N., in Hematopoietic Cell Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. 10, Golde et al., eds. [New York, Academy Press] pp 37-52 [1978]), one of which is interleukin-3 (IL-3), a multilineage colony stimulating factor (multi-CSF [Burstein, S. A., Blood Cells 11:469 [1986]). The other factors in this medium have not yet been identified and isolated. WEHI-3 is a murine myelomonocytic cell line secreting relatively large amounts of IL-3 and smaller amounts of GM-CSF. IL-3 has been recently purified and cloned (Ihle et al., J. Immunol. 129:2431 [1982]) and has been found to potentiate the growth of a wide range of hemopoietic cells (Ihle et al., J. Immunol. 13:282 [1983]). IL-3 has also been found to synergize with many of the known hemopoietic hormones or growth factors (Bartelmez et al., J. Cell Physiol. 122:362-369 [1985] and Warren et al., Cell 46:667-674 [1988]), including both erythropoietin (EPO) and H-1 (later known as interleukin-1 or IL-1), in the induction of very early multipotential precursors and the formation of very large mixed hemopoietic colonies.
Other sources of megakaryocyte potentiators have been found in the conditioned media of murine lung, bone, macrophage cell lines, peritoneal exudate cells and human embryonic kidney cells. Despite certain conflicting data (Mazur, E., Exp. Hematol. 15:340-350 [1987]), there is some evidence (Geissler et al., Br. J. Haematol. 60:233-238 [1985]) that activated T lymphocytes rather than monocytes play an enhancing role in megakaryocytopoiesis. These findings suggest that activated T-lymphocyte secretions such as interleukins may be regulatory factors in meg development (Geissler et al., Exp. Hematol. 15:845-853 [1987]). A number of studies on megakaryocytopoiesis with purified EPO (Vainchenker et al., Blood 54:940 [1979]; McLeod et al., Nature 261:492-4 [1976]; and Williams et al., Exp. Hematol. 12:734 [1984]) indicate that this hormone has an enhancing effect on meg colony formation. More recently this has been demonstrated in both serum-free and serum-containing cultures and in the absence of accessory cells (Williams et al., Exp. Hematol. 12:734 [1984]). EPO was postulated to be involved more in the single and two-cell stage aspects of megakaryocytopoiesis as opposed to the effect of PWM-SpCM which was involved in the four-cell stage of megakaryocyte development. The interaction of all these factors on both early and late phases of megakaryocyte development remains to be elucidated.
Other documents of interest include: Eppstein et al., U.S. Pat. No. 4,962,091; Chong, U.S. Pat. No. 4,879,111; Fernandes et al., U.S. Pat. No. 4,604,377; Wissler et al., U.S. Pat. No. 4,512,971; Gottlieb, U.S. Pat. No. 4,468,379; Kogan et al., U.S. Pat. No. 5,250,732; Kimura et al., Eur. J. Immunol., 20(9): 1927-1931 (1990); Secor, W. E. et al., J. of Immunol., 144(4): 1484-1489 (1990); Warren, D. J., et al., J. of Immunol. 140(1): 94-99 (1988); Warren, M. K. et al., Exp. Hematol., 17(11): 1095-1099 (1989); Bruno, E., et al., Exp. Hematol., 17(10): 1038-1043 (1989); Tanikawa et al., Exp. Hematol., 17(8): 883-888 (1989); Koike et al., Blood, 75(12): 2286-2291 (1990); Lotem, et al., Blood, 75(5): 1545-1551 (1989); Rennick, D., et al., Blood, 73(7): 1828-1835 (1989); and Clutterbuck, E. J., et al., Blood, 73(6): 1504-1512 (1989).
II. Thrombocytopenia
Platelets are critical elements of the blood clotting mechanism. Depletion of the circulating level of platelets, called thrombocytopenia, occurs in various clinical conditions and disorders. Thrombocytopenia is commonly defined as a platelet count below 150xc3x97109 per liter. The major causes of thrombocytopenia can be broadly divided into three categories on the basis of platelet life span, namely; (1) impaired production of platelets by the bone marrow, (2) platelet sequestration in the spleen (splenomegaly), or (3) increased destruction of platelets in the peripheral circulation (e.g. autoimmune thrombocytopenia or chemo- and radiation therapy). Additionally, in patients receiving large volumes of rapidly administered platelet-poor blood products, thrombocytopenia may develop due to dilution.
The clinical bleeding manifestations of thrombocytopenia depend on the severity of thrombocytopenia, its cause, and possible associated coagulation defects. In general, patients with platelet counts between 20 and 100xc3x97109 per liter are at risk of excessive posttraumatic bleeding, while those with platelet counts below 20xc3x97109 per liter may bleed spontaneously. These latter patients are candidates for platelet transfusion with attendant immune and viral risk. For any given degree of thrombocytopenia, bleeding tends to be more severe when the cause is decreased production rather than increased destruction of platelets; in the latter situation, accelerated platelet turnover results in the circulation of younger, larger and hemostatically more effective platelets. Thrombocytopenia may result from a variety of disorders briefly described below. A more detailed description may be found in Schafner, A. I., Thrombocytopenia and Disorders of Platelet Function, Internal Medicine, 3rd Ed., John J. Hutton et al., Eds., Little Brown and Co., Boston/Toronto/London, (1990).
(a) Thrombocytopenia Due to Impaired Platelet Production
Causes of congenital thrombocytopenia include constitutional aplastic anemia (Fanconi syndrome) and congenital amegakaryocytic thrombocytopenia, which may be associated with skeletal malformations. Acquired disorders of platelet production are caused by either hypoplasia of megakaryocytes or ineffective thrombopoiesis. Megakaryocytic hypoplasia can result from a variety of conditions, including marrow aplasia (including idiopathic forms or myelosuppression by chemotherapeutic agents or radiation therapy), myelfibrosis, leukemia, and invasion of the bone marrow by metastatic tumor or granulomas. In some situations, toxins, infectious agents, or drugs may interfere with thrombopoiesis relatively selectively; examples include transient thrombocytopenias caused by alcohol and certain viral infections and mild thrombocytopenia associated with the administration of thiazide diuretics. Finally, ineffective thrombopoiesis secondary to megaloblastic processes (folate or B12 deficiency) can also cause thrombocytopenia, usually with coexisting anemia and leukopenia.
Current treatment of thrombocytopenias due to decreased platelet production depends on identification and reversal of the underlying cause of the bone marrow failure. Platelet transfusions are usually reserved for patients with serious bleeding complications or for coverage during surgical procedures, since isoimmunization may lead to refractoriness to further platelet transfusions. Mucosal bleeding resulting from severe thrombocytopenia may be ameliorated by the oral or intravenous administration of the antifibrinolytic agents. Thrombotic complications may develop, however, if antifibrinolytic agents are used in patients with disseminated intravascular coagulation (DIC).
(b) Thrombocytopenia Due to Splenic Sequestration
Splenomegaly due to any cause may be associated with mild to moderate thrombocytopenia. This is a largely passive process (hypersplenism) of splenic platelet sequestration, in contrast to the active destruction of platelets by the spleen in cases of immunomediated thrombocytopenia discussed below. Although the most common cause of hypersplenism is congestive splenomegaly from portal hypertension due to alcoholic cirrhosis, other forms of congestive, infiltrative, or lymphoproliferative splenomegaly are also associated with thrombocytopenia. Platelet counts generally do not fall below 50xc3x97109 per liter as a result of hypersplenism alone.
(c) Thrombocytopenia Due to Nonimmune-mediated Platelet Destruction
Thrombocytopenia can result from the accelerated destruction of platelets by various nonimmunologic processes. Disorders of this type include disseminated intravascular coagulation, prosthetic intravascular devices, extracorporeal circulation of the blood, and thrombotic microangiopathies such as thrombotic thrombocytic purpura. In all of these situations, circulating platelets that are exposed to either artificial surfaces or abnormal vascular intima either are consumed at these sites or are damaged and then prematurely cleared by the reticuloendothelial system. Disease states or disorders in which disseminated intravascular coagulation (DIC) may arise are set forth in greater detail in Braunwald et al: (eds), Harrison""s Principles of Internal Medicine, 11th Ed., p.1478, McGraw Hill (1987). Intravascular prosthetic devices, including cardiac valves and intra-aortic balloons can cause a mild to moderate destructive thrombocytopenia and transient thrombocytopenia in patients undergoing cardiopulmonary bypass or hemodialysis may result from consumption or damage of platelets in the extracorporeal circuit.
(d) Drug-induced Immune Thrombocytopenia
More than 100 drugs have been implicated in immunologically mediated thrombocytopenia. However, only quinidine, quinine, gold, sulfonamides, cephalothin, and heparin have been well characterized. Drug-induced thrombocytopenia is frequently very severe and typically occurs precipitously within days while patients are taking the sensitizing medication.
(e) Immune (Autoimmune) Thrombocytopenic Purpura (ITP)
ITP in adults is a chronic disease characterized by autoimmune platelet destruction. The autoantibody is usually IgG although other immunoglobulins have also been reported. Although the autoantibody of ITP has been found to be associated with platelet membrane GPIIbIIIa, the platelet antigen specificity has not been identified in most cases. Extravascular destruction of sensitized platelets occurs in the reticuloendothelial system of the spleen and liver. Although over one-half of all cases of ITP are idiopathic, many patients have underlying rheumatic or autoimmune diseases (e.g. systemic lupus erythematosus) or lymphoproliferative disorders (e.g. chronic lymphocytic leukemia).
(f) HIV-Induced ITP
ITP is an increasingly common complication of HIV infection (Morris et al., Ann. Intern. Med., 96: 714-717 [1982]), and can occur at any stage of the disease progression, both in patients diagnosed with the Acquired Immune Deficiency Syndrome (AIDS), those with AIDS-related complex, and those with HIV infection but without AIDS symptoms. HIV infection is a transmissible disease ultimately characterized by a profound deficiency of cellular immune function as well as the occurrence of opportunistic infection and malignancy. The primary immunologic abnormality resulting from infection by HIV is the progressive depletion and functional impairment of T lymphocytes expressing the CD4 cell surface glycoprotein (H. Lane et al., Ann. Rev. Immunol., 3:477 [1985]). The loss of CD4 helper/inducer T cell function probably underlies the profound defects in cellular and humoral immunity leading to the opportunistic infections and malignancies characteristic of AIDS (H. Lane supra).
Although the mechanism of HIV-associated ITP is unknown, it is believed to be different from the mechanism of ITP not associated with HIV infection. (Walsh et al., N. Eng. J. Med., 311: 635-639 [1984]; and Ratner, L., Am. J. Med., 86: 194-1981 [1989]).
III. Therapy
The therapeutic approach to the treatment of patients with HlV-induced ITP is dictated by the severity and urgency of the clinical situation. The treatment is similar for HIV-associated and non-HIV-related ITP, and although a number of different therapeutic approaches have been used, the therapy remains controversial.
Platelet counts in patients diagnosed with ITP have been successfully increased by glucocorticoid (e.g. prednisolone) therapy, however in most patients the response is incomplete, or relapse occurs when the glucocorticoid dose is reduced or its administration is discontinued. Based upon studies with patients having HIV-associated ITP, some investigators have suggested that glucocorticoid therapy may result in predisposition to AIDS. Glucocorticoids are usually administered if platelet count falls below 20xc3x97109/liter or when spontaneous bleeding occurs.
For patients refractory to glucocorticoids, the compound 4-(2-chlorphenyl)-9-methyl-2-[3-(4-morpholinyl)-3-propanon-1-yl]6H-thieno[3,2,f][1,2,4]triazolo[4,3,a,][1,4] diazepin (WEB 2086) has been successfully used to treat a severe case of non HIV-associated ITP. A patient having platelet counts of 37,000-58,000/xcexcl was treated with WEB 2086 and after 1-2 weeks treatment platelet counts increased to 140,000-190,000/xcexcl. (EP 0361077A2 and Lohman, H., et al., Lancet:1147 [1988]).
Although the optimal treatment for acquired amegacaryocytic thrombocytopenia purpura (AATP) is uncertain, antithymocyte globulin (ATG), a horse antiserum to human thymus tissue, has been shown to produce prolonged complete remission (Trimble, M. S., et al., Am. J. Hematol.,37: 126-127 [1991]). A recent report however, indicates that the hematopoietic effects of ATG are attributable to thimerosal, where presumably the protein acts as a mercury carrier (Panella, T. J., and Huang, A. T., Cancer Research:50: 4429-4435 [1990]).
Good results have been reported with splenectomy. Splenectomy removes the major site of platelet destruction and a major source of autoantibody production in many patients. This procedure results in prolonged treatment-free remissions in a large number of patients. However, since surgical procedures are generally to be avoided in immune compromised patients, splenectomy is recommended only in severe cases of HIV-associated ITP, in patients who fail to respond to 2 to 3 weeks of glucocorticoid treatment, or do not achieve sustained response after discontinuation of glucocorticoid administration. Based upon current scientific knowledge, it is unclear whether splenectomy predisposes patients to AIDS.
In addition to prednisolone therapy and splenectomy, certain cytotoxic agents, e.g. vincristine, and azidothimidine (AZT, zidovudine) also show promise in treating HIV-induced ITP however, the results are preliminary.
It will be appreciated from the foregoing that one way to treat thrombocytopenia would be to obtain an agent capable of accelerating the differentiation and maturation of megakaryocytes or precursors thereof into the platelet-producing form. Considerable efforts have been expended on identifying such an agent, commonly referred to as xe2x80x9cthrombopoeitinxe2x80x9d (TPO). Thrombopoeitin activity was observed as early as 1959 (Rak et al., Med. Exp.1:125) and attempts to characterize and purify this agent have continued to the present day. While reports of partial purification of thrombopoeitin-active polypeptides exist (see, for example, Tayrien et al., J. Biol. Chem. 262:3262 [1987] and Hoffman et al., J. Clin. Invest. 75:1174 [1985]), others have postulated that thrombopoeitin is not a discrete entity in its own right but rather is simply the polyfunctional manifestation of a known hormone (IL-3, Sparrow et al., Prog. Clin. Biol. Res.,215:123 [1986]). Regardless of its form or origin, a molecule possessing thrombopoetic activity would be of significant therapeutic value. Although no protein has been unambiguously identified as thrombopoeitin, considerable interest surrounds the recent discovery that mpl, a putative cytokine receptor, may transduce a thrombopoietic signal.
IV. Mpl is a Cytokine Receptor
It is believed that the proliferation and maturation of hematopoietic cells is tightly regulated by factors that positively or negatively modulate pluripotential stem cell proliferation and multilineage differentiation. These effects are mediated through the high-affinity binding of extracellular protein factors to specific cell surface receptors. These cell surface receptors share considerable homology and are generally classified as members of the cytokine receptor superfamily. Members of the superfamily include receptors for: IL-2 (beta and gamma chains) (Hatakeyama et al., Science 244:551-556 [1989]; Takeshita et al., Science 257:379-382 [1991]), IL-3 (Itoh et al., Science 247:324-328 [1990]; Gorman et al., Proc. Natl. Acad. Sci. USA 87:5459-5463 [1990]; Kitamura et al., Cell 66:1165-1174 [1991a]; Kitamura et al. Proc. Natl. Acad. Sci. USA 88:5082-5086 [1991b]), IL-4 (Mosley et al., Cell 59:335-348 [1989], IL-5 (Takaki et al. EMBO J. 9:4367-4374 [1990]; Tavernier et al., Cell 66:1175-1184 [1991]), IL-6 (Yamasaki et al., Science 241:825-828 [1988]; Hibi et al. Cell 63:1149-1157 [1990]), IL-7 (Goodwin et al. Cell 60:941-951 [1990]), IL-9 (Renault et al. Proc. Natl. Acad. Sci. USA 89:5690-5694 [1992]), granulocyte-macrophage colony-stimulating factor (GM-CSF) (Gearing et al., EMBO J. 8:3667-3676 [1991]; Hayashida et al. Proc. Natl. Acad. Sci. USA 244:9655-9659 [1990]), granulocyte colony-stimulating factor (G-CSF) (Fukunaga et al., Cell 61:341-350 [1990a]; Fukunaga et al. Proc. Natl. Acad. Sci. USA 87:8702-8706 [1990b]; Larsen et al. J. Exp. Med. 172:1559-1570 [1990]), erythropoietin (EPO) (D""Andrea et al., Cell 57:277-285 [1989]; Jones et al., Blood 76:31-35 [1990]), Leukemia inhibitory factor (LIF) (Gearing et al., EMBO J. 10:2839-2848 [1991]), oncostatin M (OSM) (Rose et al., Proc. Natl. Acad. Sci. USA 88:8641-8645 [1991]) and also receptors for prolactin (Boutin et al., Proc. Natl. Acad. Sci. USA 88:7744-7748 [1988]; Edery et al., Proc. Natl. Acad. Sci. USA 86:2112-2116 [1989]), growth hormone (GH) (Leung et al., Nature 330:537-543 [1987]) and ciliary neurotrophicfactor (CNTF) (Davis et al., Science 253:59-63 [1991].
Members of the cytokine receptor superfamily may be grouped into three functional categories (for review see Nicola et al., Cell 67:1-4 [1991]). The first class comprises single chain receptors, such as erythropoietin receptor (EPO-R) or granulocyte colony stimulating factor receptor (G-CSF-R) bind ligand with high affinity via the extracellular domain and also generate an intracellular signal. A second class of receptors, so call xcex1-subunits, includes interleukin-6 receptor (IL6-R), granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R), interleukin-3 receptor (IL3-Rxcex1) and other members of the cytpkine receptor superfamily. These xcex1-subunits bind ligand with low affinity but cannot transduce an intracellular signal. A high affinity receptor capable of signaling is generated by a heterodimer between an xcex1-subunit and a member of a third class of cytokine receptors, termed xcex2-subunits, e.g. xcex2c, the common xcex2-subunit for the three xcex1-subunits IL3-Rxcex1 and GM-CSF-R.
Evidence that mpl is a member of the cytokine receptor superfamily comes from sequence homology (Gearing, D. P., EMBO J. 8:3667-3676 [1988]; Bazan, J. F., Proc. Natl. Acad. Sci. USA 87:6834-6938 [1990]; Davis S., et al., Science 253:59-63 [1991] and Vigon et al., Proc. Natl. Acad. Sci. USA 89:5640-5644 [1992]) and its ability to transduce proliferative signals.
Deduced protein sequence from molecular cloning of murine c-mpl reveals this protein is homologous to other cytokine receptors. The extracellular domain contains 465 amino acid residues and is composed of two subdomains each with four highly conserved cysteines and a WGXWS motif in the N-terminal subdomain and WSXWS motif in the C-terminal subdomain. The ligand-binding extracellular domains are predicted to have similar double xcex2-barrel fold structural geometries. This duplicated extracellular domain is highly homologous to the signal transducing chain common to IL-3, IL-5 and GM-CSF receptors as well as the low-affinity binding domain of LIF (Vigon I., et al., Oncogene 8:2607-2615 [1993]). Thus mpl may belong to the low affinity ligand binding class of cytokine receptors.
The extracellular domain is followed by a 22 residue transmembrane domain and a 121 residue cytoplasmic domain rich in serine and proline. The cytoplasmic domain contains no consensus protein kinase, phosphotase or any other known motif associated with signal transduction.
A comparison of murine mpl and mature human mpl P, reveals these two proteins show 81% sequence identity. More specifically, the N-terminus and C-terminus extracellular subdomains share 75% and 80% identity respectively. The most conserved mpl region is the cytoplasmic domain showing 91% amino acid identity, with a sequence of 37 residues near the transmembrane domain being identical in both species. Accordingly, mpl is reported to be one of the most conserved members of the cytokine receptor superfamily (Vigon supra).
Evidence that mpl is a functional receptor capable of transducing a proliferative signal comes from construction of chimeric receptors containing an extracellular domain from a cytokine receptor having high affinity for a known cytokine with the mpl cytoplasmic domain. Since no known ligand for mpl has been reported, it was necessary to construct the chimeric high affinity ligand binding extracellular domain from a class one cytokine receptor such as IL-4R or G-CSFR. Vigon et al. supra fused the extracellular domain of G-CSFR with both the transmembrane and cytoplasmic domain of c-mpl. An IL-3 dependent cell line, BAF/B03 was transfected with the G-CSFR/mpl chimera along with a full length G-CSFR control. Cells transfected with the chimera grew equally well in the presence of cytokine IL-3 or G-CSF. Similarly, cells transfected with G-CSFR also grew well in either IL-3 or G-CSF. All cells died in the absence of growth factors. A similar experiment was conducted by Skoda, R. C. et al. EMBO J. 12(7):2645-2653 (1993) in which both the extracellular and transmrembrane domains of human IL4 receptor (hIL4-R) were fused to the murine mpl cytoplasmic domain, and transfected into a murine IL3 dependent Ba/F3 cell line. Ba/F3 cells transfected with wildtype hIL4-R proliferated normally in either the presence of the species specific IL-4 or IL-3. BaF3 cells transfected with hIL4R/mpl proliferated normally in the presence of hIL4 (in the presence or absence of IL3) demonstrating that in Ba/F3 cells the mpl cytoplasmic domain contains all the elements necessary to transduce a proliferative signal.
These chimeric experiments demonstrate the proliferation signaling capability of the mpl extracellular domain but are silent regarding whether the mpl extracellular domain can bind a ligand. These results are consistent with at least two possibilities, namely, mpl is a single chain (class one) receptor like EpoR or G-CSFR or it is a signal transducing xcex2-subunit (class three) requiring an xcex1-subunit like IL-3 (Skoda et al. supra).
V. Mpl Ligand Stimulates Megakaryocytopoiesis
As described above, it has been suggested that serum contains a unique factor, sometimes referred to as thrombopoietin, that acts synergistically with various other cytokines to promote growth and maturation of megakaryocytes. No such natural factor has ever been isolated from serum or any other source even though considerable effort has been expended by numerous groups. Even though it is not know whether mpl is capable of directly binding a megakaryocyte stimulating factor, recent experiments demonstrate that mpl is involved in proliferative signal transduction from a factor or factors found in the serum of patients with aplastic bone marrow (Methia, N. et al., Blood 82(5):1395-1401 [1993]).
Evidence that a unique serum colony-forming factor distinct from IL-1xcex1, IL-3, IL-4, IL-6, IL-11, SCF, EPO, G-CSF, and GM-CSF transduces a proliferative signal through mpl comes from examination of the distribution of c-mpl expression in primitive and committed hemotopoietic cell lines and from mpl antisense studies in one of these cell lines.
Using reverse transcriptase (RT)-PCR in immuno-purified, human hematopoietic cells Methia et al. supra demonstrated that strong mpl mRNA messages were only found in CD34+ purified cells, megakaryocytes and platelets. CD34+ cells purified from Bone Marrow (BM) represents about 1% of all BM cells and are enriched in primitive and committed progenitors of all lineages (e.g. erythroid, granulomacrophage, and megakaryocytic).
Mpl antisense oligodeoxy nucleotides were shown to suppress megakaryocytic colony formation from the pluripotent CD34+ cells cultured in serum from patients with aplastic marrow (a rich source of megakaryocytes stimulating activity [Meg-CSA]). These same antisense oligodeoxynucleotides had no effect on erythroid or granulomacrophage colony formation.
Whether mpl directly binds a ligand and whether the serum factor shown to cause megakaryocytopoiesis acts through mpl is still unknown. It has been suggested, however, that if mpl does directly bind a ligand, its amino acid sequence is likely to be highly conserved and have species cross reactivity owing to the considerable sequence identity between human and murine mpl extracellular domain (Vigon et al., supra[1993]).
In view of the foregoing it will be appreciated there is a current and continuing need in the art to isolate and identify molecules capable of stimulating differentiation and maturation of megakaryocytes for therapeutic use in the treatment of thrombocytopenia. It is believed such a molecule is a mpl ligand and thus there exists a further need to isolate such ligand(s) to evaluate their role(s) in cell growth and differentiation.
Accordingly, it is an object of this invention to obtain a novel pharmaceutically pure molecule capable of stimulating differentiation and maturation of megakaryocytes into the mature platelet-producing form.
It is another object to provide the novel molecule in a form for therapeutic use in the treatment of thrombocytopenia.
It is a further object of the present invention to isolate, purify and specifically identify novel protein ligands capable of binding in vivo a cytokine superfamily receptor known as mpl and to transduce a proliferative signal.
It is still another object to provide novel nucleic acid molecules encoding such protein ligands and to use this nucleic acid molecule to produce mpl binding ligands in recombinant cell culture for diagnostic and therapeutic use.
It is yet another object to provide derivatives and modified forms of the protein ligands including amino acid sequence variants, novel glycoprotein forms and other covalent derivatives thereof.
It is an additional object to provide fusion polypeptide forms combining a novel mpl ligand and a heterologous protein and covalent derivatives thereof.
It is yet an additional object to prepare immunogens for raising antibodies against novel mpl ligands or fusion forms thereof, as well as to obtain antibodies capable of binding such ligands.
These and other objects of the invention will be apparent to the ordinary artisan upon consideration of the specification as a whole.
The objects of the invention are achieved by providing an isolated mammalian megakaryocytopoietic maturation promoting protein capable of stimulating maturation and/or differentiation of megakaryocytes into the mature platelet-producing form. This substantially homogeneous protein denominated the xe2x80x9cmpl ligandxe2x80x9d is purified from a natural source by the procedures described herein and has the following characteristics:
(1) The partially purified ligand isolated from aplastic porcine plasma elutes from a gel filtration run in either PBS, PBS containing 0.1% SDS or PBS containing 4M MgCl2 with Mr of 60,000-70,000;
(2) The ligand""s activity is destroyed by pronase;
(3) The ligand is stable to low pH (2.5), SDS to 0.1%, and 2M urea;
(4) The ligand is a glycoprotein, based on its binding to a variety of lectin columns;
(5) The highly purified ligand elutes from non-reduced SDS-PAGE with a Mr of 25,000-35,000. Smaller amounts of activity also elute with Mr of xcx9c18,000 and 60,000;
(6) The highly purified ligand resolves on reduced SDS-PAGE as a doublet with Mr of 28,000 and 31,000;
(7) The amino-terminal sequence of the 18,000, 28,000 and 31,000 bands is the samexe2x80x94SPAPPACDPRLLNKLLRDDH SEQ ID NO.: 1); and
(8) The ligand binds and elutes from the following affinity columns
Blue-Sepharose,
CM Blue-Sepharose,
MONO-Q,
MONO-S,
Lentil lectin-Sepharose,
WGA-Sepharose,
CON A-Sepharose,
Ether 650 m Toyopearl,
Butyl 650 m Toyopearl,
Phenyl 650 m Toyopearl, and
Phenyl-Sepharose.
The xe2x80x9cmpl ligandxe2x80x9d polypeptide of this invention preferably has at least 80% sequence identity with the amino acid sequence of the highly purified mpl ligand isolated from aplastic porcine plasma described herein. Most preferably the mpl ligand of this invention is mature human mpl ligand or a protein having 80% sequence identity with mature human mpl ligand. Optionally the mpl ligand polypeptide or fragment thereof may be fused to a heterologous polypeptide. A preferred heterologous polypeptide is a cytokine.
Another aspect of this invention provides a composition comprising an isolated mpl ligand capable of stimulating the incorporation of labeled nucleotides (3H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with human mpl P and isolated from its source environment sufficiently free of contaminating source proteins to yield an unambiguous N-terminus amino acid sequence of at least 20 residues. In one embodiment, the N-terminus amino acid sequence of this ligand is SPAPPACDPRLLNKLLRDDH (SEQ ID NO.: 1). Optionally, the ligand of this invention has a portion of its amino acid sequence at or near its N-terminus that has at least 80% sequence identity with the sequence; SPAPPACDPRLLNKLLRDDH (SEQ ID NO.:1).
In still another aspect, a method is provided for purifying mpl ligand molecules comprising contacting a source plasma containing the mpl ligand molecules to be purified with an immobilized receptor polypeptide, specifically mpl or a mpl fusion polypeptide immobilized on a support, under conditions whereby the mpl ligand molecules to be purified are selectively adsorbed onto the immobilized receptor polypeptide, washing the immobilized support to remove non-adsorbed material, and eluting the molecules to be purified from the immobilized receptor polypeptide to which they are adsorbed with an elution buffer. Preferably the source plasma containing the mpl ligand is aplastic porcine plasma and the immobilized receptor is a mpl-IgG fusion. Also preferably the immobilized support is washed with PBS/PBS in 2M NaCl and the elution buffer is 0.1M glycine-HCl, pH 2.25.
In another embodiment, this invention provides an isolated antibody capable of binding to the mpl ligand. The isolated antibody capable of binding to the mpl ligand may optionally be fused to a second polypeptide and the antibody or fusion thereof may be used to isolate and purify mpl ligand from a source as described above for immobilized mpl polypeptide. In a further aspect of this embodiment, the invention provides a method for detecting the mpl ligand in vitro or in vivo comprising contacting the antibody with a sample, especially a serum sample, suspected of containing the ligand and detecting if binding has occurred.
In still further embodiments, the invention provides an isolated nucleic acid molecule, encoding the mpl ligand or fragments thereof, which nucleic acid molecule may be labeled or with a detectable moiety, and a nucleic acid molecule having a sequence that is complementary to, or hybridizes under stringent or moderately stringent conditions with, a nucleic acid molecule having a sequence encoding a mpl ligand. In a further aspect of this embodiment, the nucleic acid molecule is DNA encoding the mpl ligand and further comprises a replicable vector in which the DNA is operably linked to control sequences recognized by a host transformed with the vector. This aspect further includes host cells transformed with the vector and a method of using the DNA to effect production of mpl ligand, comprising expressing the DNA encoding the mpl ligand in a culture of the transformed host cells and recovering the mpl ligand from the host cells or the host cell culture. The mpl ligand prepared in this manner is preferably human mpl ligand.
The invention further includes a method for treating a mammal having thrombocytopenia comprising administering a therapeutically effective amount of a mpl Ligand to the mammal. Optionally the mpl Ligand is administered in combination with a cytokine, especially a colony stimulating factor or interleukin. Preferred colony stimulating factors or interleukins include; G-CSF, CSF-1, GM-CSF, M-CSF, erythropoietin, IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, or IL-11.